Measurement of small molecule analytes present in blood samples is important for understanding the health status of a subject; however, many small molecules are bound by blood proteins and other molecules that interfere with such measurements. Enzymes have been used in attempts to free target analytes from binding proteins; for example, Bates used pepsin treatment (at pH 1) to free thyroxine from serum proteins for radioimmunoassay measurements in serum following centrifugation of an acid-treated sample (U.S. Pat. No. 3,962,039). However, such harsh treatment of a sample and the use of radioactive tracers to identify analytes are problematic.
Vitamin D is a steroid vitamin essential for normal calcium homeostasis, and thus important, for example, in bone health. Vitamin D comes in many forms; e.g., vitamin D1 through vitamin D5. Two forms of vitamin D are important in humans: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). In addition to these forms of vitamin D, hydroxy forms of the vitamin may also be found circulating in the blood of a human subject; for example, common hydroxyl forms of vitamin D2 include 25-hydroxy vitamin D2 and 1,25-dihydroxy vitamin D2. Common hydroxyl forms of vitamin D3 include 25-hydroxy vitamin D3 and 1,25-dihydroxy vitamin D3. The major circulating forms of vitamin D in blood are the 25-hydroxyl forms of vitamin D (25-hydroxy vitamin D3 and 25-hydroxy vitamin D2).
Vitamin D3 is formed in the skin from its precursor 7-dehydrocholesterol after ultraviolet irradiation or is absorbed from the diet. It is further hydroxylated in the liver to 25-hydroxy vitamin D3 as the first step of its conversion in the kidney to 1,25-dihydroxyvitamin D3, which is the biologically active form. 25-hydroxy vitamin D3 is the main circulating form of vitamin D, but 25-hydroxy vitamin D2 is also found especially in subjects who take certain vitamin supplements. So it is important to measure both forms of 25-hydroxyvitamin D when monitoring vitamin D status and the effect of vitamin D2 supplementation on vitamin D status. Vitamin-D (in all its isoforms) is bound tightly to Vitamin-D binding protein (VDBP) in blood. In order to measure vitamin D levels in blood, it has to be extracted or displaced from the binding protein. Extraction methods are typically slow and cumbersome.
Armbruster et al. (U.S. Pat. No. 7,964,363) discuss a method for measuring vitamin D levels in a blood sample at neutral pH using a serine protease with endo- and exoproteolytic activity (e.g., proteinase K, Enzyme Commission (EC) number EC 3.4.21.64) in order to digest vitamin D binding proteins in blood plasma or serum; the serine protease is inactivated by addition of a dilution buffer, which allows subsequent use of a monoclonal antibody in determining vitamin D from the serum or plasma sample. However, dilution will reduce the concentration of the protease, rather than inactivate the protease, or may require excessive dilution which also dilutes the sample and the analyte, rendering measurement of the analyte more difficult.
A method for measuring blood levels of vitamin D that has become standard is a radioimmunoassay (RIA) method using a 125I-labeled vitamin D tracer (Hollis et al., Clinical Chemistry 39:529-533 (1993)), available commercially as the DiaSorin RIA33 test using methods and equipment from Diasorin Corporation (Stillwater, Minn.). This RIA method uses acetonitrile extraction followed by competitive radioimmunoassay using 125I-labelled 25-hydroxy vitamin D and an antibody to 25-hydroxy vitamin D. A second antibody is used as precipitating agent. A different assay, the Diasorin Liaison® test, which does not use radioactive tracers was also developed by the Diasorin Corporation. The Diasorin Liaison® test is a chemiluminescent assay in which serum is incubated with antivitamin-D coated microparticles and isoluminol derivative-conjugated 25-hydroxy vitamin D before measurement of the chemiluminescent signal. Other common vitamin D assays include the IDS Gamma-B assay (which also uses acetonitrile extraction to release vitamin D for detection by antibodies) (ImmunoDiagnostic Systems (IDS) Ltd., Scottsdale, Ariz., USA), and the Nichols Advantage assay (which uses a denaturing agent to separate 25-hydroxy vitamin D from its binding protein) (Nichols Institute Diagnostics, San Clement, Calif.).
However, comparisons of various vitamin D assays, including separate measurements of vitamin D2 and vitamin D3, indicate that the results obtained by different methodologies, and in some cases, by different laboratories, do not always agree (Binkley et al., J Clin Endocrinol Metab 89:3152-3157 (2004); Hollis, Clinical Chemistry 46(10): 1657-1661 (2000); Lensmeyer et al., Clinical Chemistry 52(6):1120-1126 (2006)). For example, reports have been published suggesting that the Nichols Advantage assay is unable to measure samples containing substantial amounts of 25-hydroxy vitamin D2 reliably (Carter et al., Clinical Chemistry 50(11):2195-2197 (2004); Terry et al., Clinical Chemistry 51(8):1565-1566 (2005)).
Vitamin-D deficiency is associated with bone disease. In its extreme form when left untreated in children the deficiency leads to osteomalacia or rickets, a severe chronic and irreversible condition. Vitamin D supplements provide benefits in the form of enhanced bone health and decreased mortality in elderly women taking such supplements. However, excess vitamin D may cause toxicity; thus, it is believed that vitamin D levels should be maintained, for example, between about 25 ng/milliliter to about 75 ng/milliliter of blood serum. Measurements of vitamin D are needed in order to identify abnormal vitamin D levels in a patient and to confirm response to therapy and the proper maintenance of normal levels.
Thus, it would be beneficial to be able to make rapid measurements of vitamin D in a sample of blood from a subject.